Methods and compositions for diagnosing and suppressing metastasis thereof

ABSTRACT

Disclosed is a method for determining metastatic potential of tumors, a method and a composition for treating/suppressing metastasis of cancers, as well as a method for obtaining a metastasis suppressor. A particular aspect of the invention relates to a method and a composition of evaluating expression levels of connective tissue growth factor (CTGF, also known as CCN2) to determine the status of tumor invasion. Another aspect of the invention relates to a method and a composition comprising a tumor suppressor, which maintains or increases an expression level of CTGF to suppress the invasion ability of tumor cells effectively. In another aspect of the invention relates to methods for obtaining a metastasis suppressor by evaluating the expression levels of CTGF after contacting suppressor candidates with a tumor cell line system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.10/871,407, filed on Jun. 18, 2004, now allowed, which claims priorityfrom Taiwanese Patent Application No. 093100208, filed on Jan. 6, 2004,all of which are hereby incorporated by reference in their entirety.

Although incorporated by reference in its entirety, no arguments ordisclaimers made in the parent application apply to this divisionalapplication. Any disclaimer that may have occurred during theprosecution of the above-referenced application(s) is hereby expresslyrescinded. Consequently, the Patent Office is asked to review the newset of claims in view of all of the prior art of record and any searchthat the Office deems appropriate.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and composition for diagnosingand suppressing metastasis thereof. A particular aspect of the inventionrelates to a method and composition of evaluating expression levels ofconnective tissue growth factor (CTGF, also known as CCN2) to determinethe status of tumor invasion. Another aspect of the invention relates toa method and composition comprising a tumor suppressor, which maintainsor increases an expression level of CTGF to suppress the invasionability of tumor cells effectively.

2. The Prior Arts

Cancer is a leading cause of death in most developed countries. Althoughmany researchers focus their efforts on developing effective methods totreat cancers, the difficulty of treating metastatic cancers is still aproblem.

Metastasis is a process that the cancer cells spread from their originalsite to other areas in the body. Tumor metastasis involves detachment oftumor cells from the primary tumor mass, microinvasion of tumor cellsinto stromal tissue, intravasation of tumor cells into blood vessels,extravasation and growth of tumor cells in secondary sites. Manyapproaches have been developed to study the metastatic process inmolecular levels, but the mechanisms of metastasis are still not veryclear.

Occurrence of metastasis may bring high risk and death rate to patients.To optimize the prognosis, some aggressive methods with severeside-effects are taken to treat or suppress the tumor metastasis. Also,early detection of primary, metastatic, and recurrent disease cansignificantly impact the prognosis of patients suffering from cancers.Accordingly, determination of metastatic potential of tumor cells isvery important to take adequate treatments to patients. Furthermore,there is a great need for methods which are effective to treat orsuppress the tumor metastasis.

SUMMARY OF THE INVENTION

The primary object of the present invention is to provide a method fordetermining metastatic potential of tumor cells. Especially, the methodrelates to evaluating expression levels of CTGF encoded by SEQ ID NO: 1,which levels are inversely associated with tumor metastasis, todetermine a metastatic potential of tumor cells. A particular aspect ofthe invention relates to determining levels of CTGF or mRNA encodingCTGF in cells of a biopsy sample to determine metastatic potential oflung or colorectal tumors. Another aspect of the invention relates todetermining levels of CT module (SEQ ID NO: 2) in a nucleotide encodingCTGF or mRNA of the CT module in cells of a sample to determine cellinvasive capacity of lung or colorectal tumors.

The secondary object of the present invention is to provide acomposition for treating or suppressing metastatic tumors, especiallylung or colorectal tumors. In one aspect of the invention, a compositionis provided, comprising a tumor suppressor, which maintains or increasesthe expression levels of CTGF to effectively suppress the invasionability of tumor cells. Particularly, the invention relates to acomposition comprising an effective amount of CTGF or a nucleotidesequence encoding CTGF for treating or suppressing metastatic tumors.

Another object of the present invention is to provide a method fortreating or suppressing metastatic tumors, especially for suppressingmetastatic lung or colorectal tumors. A particular aspect of theinvention relates to a method for treating or suppressing metastatictumors by administrating a pharmaceutical composition in an effectiveamount to an individual suffering from a cancer, which compositioncomprises a tumor suppressor that maintains or increases an expressionlevel of CTGF to suppress the invasion ability of tumor cellseffectively. Especially, in this regard, the invention relates to amethod using CTGF or a nucleotide sequence encoding CTGF to treat orsuppress metastasis of lung or colorectal tumors. Further, this aspectof the invention may be accomplished by genetic therapy.

A further object of the present invention is to provide a method forobtaining a metastasis suppressor to elevate expression levels of CTGF.This aspect of the invention particularly relates to screening out andobtaining a metastasis suppressor by evaluating the ability of elevatingexpression levels of CTGF.

In some embodiments of the invention, tissue and fluid samples may beexamined to evaluate expression levels of CTGF to determine themetastatic potential of the cells in the samples. Conventional methodssuch as immunoassays, ELISA assays, Western blots orimmunohistochemistry may be performed to evaluate CTGF levels insamples. Alternatively, levels of mRNA encoding CTGF may be determinedto evaluate the expression levels of CTGE The mRNA encoding CTGF or cDNAgenerated therefrom can be determined using methods such as PCRamplification.

In accordance with one object of the invention, a composition fortreating or suppressing metastatic tumors, comprises a suppressor thatmaintains or increases the expression levels of CTGF to effectivelysuppress the invasion ability of tumor cells. Preferably, CTGF or anucleotide sequence encoding CTGF is used as the tumor suppressor in thecomposition. In some embodiments, the composition may comprise othertypes of active ingredients or pharmaceutically acceptable carrier incombination with the suppressor.

In accordance with another object of the invention, a method fortreating or suppressing metastatic tumors comprises the steps ofadministrating a pharmaceutical composition to an individual sufferingfrom a cancer, which composition comprises an effective amount of tumorsuppressor maintaining or increasing the expression levels of CTGF,thereby effectively suppressing the tumor metastasis. In a preferredembodiment, the tumor suppressor is CTGF or a nucleotide sequenceencoding CTGF.

In accordance with another object of the invention, a method forobtaining a metastasis suppressor by elevating expression levels of CTGFcomprises the steps of (1) providing a system with a tumor cell line;(2) contacting a tumor suppressor candidate with the system; (3)evaluating the expression levels of CTGF in the tumor cells; and (4)screening out the tumor suppressors that elevates expression levels ofCTGF in tumor cells. Preferably, the tumor cell line is selected from agroup consisting of lung and colorectal cancer cell lines. Morepreferably, the tumor cell line is selected from lung or colorectalcancer cell lines with high invasiveness.

Having been briefly described, the present invention will be furtherexplained with examples and figures illustrating its practice set forthbelow. These examples and figures should not, however, be considered tolimit the scope of the invention, which is defined by the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The related figures in connection with the detailed description of thepresent invention to be made later are described briefly as follows, inwhich:

FIG. 1 shows expression of CTGF and in vitro invasion ability in humanlung cancer cell lines. A (Upper left panel): Western blot analysis ofthe expression of CTGF and cysteine-rich 61(Cyr61) proteins. Totalproteins are extracted from CL1-0, CL1-3, and CL1-5 cells and probedwith polyclonal antibody specific for CTGF or Cyr61. Each lane contains40 μg of total protein. β-Actin is used as an internal loading control.A (Upper right panel): Reverse transcription-polymerase chain reactionanalysis (RT-PCR) for CTGF and Cyr61 mRNAs. A β-actin probe is used asan internal control for RNA quantity. A (Lower): The invasion activityof each clone is measured in vitro with the Boyden chamber after 48hours. Each cell subline is assayed in three experiments carried out intriplicate. Error bars correspond to 95% confidence intervals. B(Upper): Western blot analysis of CTGF and Cry61 proteins in these humanlung adenocarcinoma cancer cell lines H928, NICH520, and A549. B(Lower): In vitro invasion activity of human lung adenocarcinoma cancercell lines is measured with the Boyden chamber after 48 hours. Theseexperiments are repeated four times. Data are the mean; error bars showthe corresponding upper 95% confidence interval.

FIG. 2 shows overexpression of CTGF and suppression of invasion abilityin transfected CL1-5 cells. A: Expression of CTGF protein inmock-transfected and CTGF-transfected CL1-5 clones assessed byimmunoblot analysis to facilitate comparison of the relative CTGFprotein expression between the mock-transfected clone CL1-5/neo and theCTGF-transfected clones CL1-5/CTGF-M, CTGF-3, and CTGF-10. B: In vitroinvasion activity of CL1-5 cells stably transfected with CTGF or controlvectors. In vitro invasion is measured in vitro with the Boyden chamberafter 48 hours by determining the percentage of cells that migratethrough Matrigel-coated filters in Transwell chambers (8-μm pore size).The invasive activity is statistically significantly lower inCTGF-transfected clones than CL1-5/neo vector control clone, asindicated by the asterisks (P=0.003, by a two-tailed Student's t test).Each clone is assayed in three experiments carried out in triplicate.

FIG. 3 shows overexpression of CTGF and suppression of invasion abilityin transfected A549 cells. Upper panel: Immunoblot analysis of CTGF anda-tubulin expression in the mock-transfected A549 (A549/neo) cells, andCTGF-overexpressed mixture clone A549/CTGF-M. Lower panel: Expression ofCTGF and the in vitro invasion activity of A549 cells stably transfectedwith CTGF or control vector. To compare the relative invasivenessbetween A549/neo and A549/CTGF-M cells, values are normalized to that ofA549/neo cells. Error bars are the corresponding upper 95% confidenceintervals. CTGF-transfected clones have statistically significantlylower invasive activity as shown by asterisks (P<0.001, by a two-tailedStudent's t test) than A549/neo clones.

FIG. 4 shows growth properties of the vector (neo) and CTGF-transfectedCL1-5 cells in monolayer culture cell lines. All experiments are carriedout in triplicate on separate occasions with similar results. Error barscorrespond to 95% confidence intervals.

FIG. 5 shows truncated CTGF expression in transfected CL1-5 cells andinvasiveness of the cells. A: Schematic representation of the CTGFfull-length and truncated proteins expressed by the pcDNA3 plasmids.Numbers above the structural motifs indicate the nucleotide position ofCTGF exon boundaries. IGFBP represents insulin-like growth factorbinding protein; VWC represents Von Willebrand-type C motif; TSP1represents thrombospondin type 1 motif; and CT representscarboxyl-terminal cysteine knots module. B: Invasive ability of CTGFdeletion mutants. Serial deletion mutants are transiently transfectedinto CL1-5 cells and then seeded into Boyden chambers for 48 hours. Eachclone is assayed in at least three experiments carried out in triplicatewith similar results. *P<0.001 versus serial deletion mutants by using atwo-tailed Student's t test.

FIG. 6 shows suppression of human lung adenocarcinoma cell metastasis byCTGF in vivo. A: 10⁶ control vector- or CTGF-transfected tumor cells(CL1-5/CTGF-M or A549/CTGF-M) are suspended in 0.1 mL of PBS andinjected into the lateral tail vein of SCID mice (10 mice per group).Animals are killed 2 months after intravenous injection, and lungs areexcised and photographed after fixation. White arrows indicatemetastatic tumor nodules. B: Histologic analysis of lung metastasis ofCL1-5/neo control cells and CL1-5/CTGF-M cells. Lungs are embedded inparaffin, paraffin-embedded tissue is sectioned (4 μm thick), andsections are stained with hematoxylin-eosin. A metastatic tumor (T) isshown within the lung parachyma of a CL1-5/neo tumor (Left). TheCL1-5/CTGF tumor (Right) has fewer foci.

FIG. 7 shows immunohistochemistry of lung cancer tissue for CTGFexpression. A: Representative staining of normal lung epithelium. Theepithelial cells (black arrow) stain at intensity level 3 for CTGF. B:Representative section of lung adenocarcinoma (stage I). Carcinomastains at intensity level 3 for CTGE C: Representative section of a lungcancer metastasis to the brain (stage VI). The tumor part stains forCTGF at intensity level 1. D: Areas with carcinoma staining negative forCTGF are surrounded by areas of stromal cells staining at intensitylevels 2 to 3 (black arrow). Scale bars represent 100 μm for A to D.

FIG. 8 shows Kaplan-Meier survival plots for patients with lungadenocarcinoma, grouped by the level of expression of CTGF protein. A:CTGF expression and disease-free survival. B: CTGF expression andoverall survival. Overall survival is assessed in 78 patients with lungadenocarcinoma in relation to CTGF expression. Solid line representspatients with high expression (levels 3 and 2) of CTGF; dotted linerepresents patients with reduced or no expression of CTGF (levels 1 and0). P value is determined by a two-sided log-rank test. The 20-month,40-month, and 60-month disease-free survival rates are 52% (95% CI is32% to 72%), 43% (95% CI is 23% to 64%), and 35% (95% CI is 15% to 54%),reapectively, for patients whose tumors express high levels of CTGF, and38% (95% CI is 25% to 52%), 30% (95% CI is 17% to 43%), and 22% (95% CIis 10% to 33%), respectively, for patients whose tumors express lowlevels of CTGF. The 20-month, 40-month, and 60-month overall survivalrates are 87% (95% CI is 74% to 100%), 75% (95% CI is 58% to 92%), and54% (95% CI is 34% to 74%), respectively, for patients whose tumorsexpress high levels of CTGF, and 48% (95% CI is 35% to 61%), 33% (95% CIis 21% to 46%), and 26% (95% CI is 14% to 38%), respectively, forpatients whose tumors express low levels of CTGF.

FIG. 9 shows CTGF expression and invasive ability of different coloncancer cell lines. A: Immunoblot analysis of CTGF and β-actin in humancolon cancer cell lines. Protein extracts (40 μg/lane) from theindicated cell lines are electrophoresed, transferred, and used forimmunodection of CTGF and β-actin (for an internal loading control)(upper). mRNA levels of CTGF and β-actin are measuredsemi-quantitatively by RT-PCR (lower). B: In vitro invasion assayperforms in human colon cancer cell lines. Approximately 2.5×10⁴ cellsin 100 μL of complete medium are placed in the upper Boyden chamber withfilter inserts (pore size, 8 μm) coated with Matrigel. After 48 hours inculture, cells are fixed in methanol and cells on the underside of thefilters are viewed and counted. Each clone is plated in triplicate ineach experiment.

FIG. 10 shows CTGF expression in control vector (Neo) (HCT116/Neo,CT26/Neo), CTGF vector (HCT116/CTGF) and CTGF-antisense vector(CT26/AS-CTGF) transfected cells and the invasion abilities of thesetransfectants. A: mRNA levels of CTGF and β-actin as detected by RT-PCR(Upper). Western blot analysis of total cell lysates probed withantibodies to CTGF and β-actin (Lower). Stable clones are selected andobtained on 800 μg/ml G418. B: Invasion ability of HCT-116/Neo,HCT-116/CTGF (Left), and CT-26/Neo, CT26/AS-CTGF (Right). Uppermicrographs show crystal violet staining of invading cells.

FIG. 11 shows liver metastasis of CT26/Neo and CT26/AS-CTGF transfectantcells. The photographs are spleens and livers from mice,which spleensare injected with either CT26/Neo or CT26/AS-CTGFcells; the livers andspleens are harvested when mice are moribund or after 6 weeks.

FIG. 12 shows histological analysis of liver metastasis of CT26/Neocells and CT26-AS-CTGF cells, as indicated. Livers are embedded inparaffin and the paraffin-embedded tissue and sectioned (4 μm thick).The sections are stained with hematoxylin-eosin. Metastatic tumors (T)are shown within the liver parachyma. The CT26/Neo tumor (upper) showsfewer foci, and a smooth margin between the normal hepatocytes. TheCT26/AS-CTGF tumor shows a satellite nodule (arrow) and an unsmooth,invaded border between the liver parachyma. The magnification factor ofthe CT26/AS-CTGF tumor is 100×.

FIG. 13 shows representative CTGF immunohistochemical staining of normalhuman colon tissue, polyp, and colorectal carcinomas. High CTGFexpression is shown in normal colon tissue (40× objective) (A) and colonpolyp (B) (100×). The left upper insert in (A) shows negative controlIgG staining of colon adenocarcinoma. High CTGF expression is found inmoderate-differentiated adenocarcinoma tissue (C). CTGF is localizeddistinctly in the apical cytoplasm and membrane of the tumor cells. LowCTGF protein expression is noted in a poor-differentiated adenocarcinomaspecimen (D).

FIG. 14 shows univariate analysis of survival (Kaplan-Meier) consideringstage II and III colorectal cancer patients. According to their CTGFexpression of tumors, overall survival and disease-free survival of 119patients are shown in (A) and (B), respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT EXAMPLE 1

Expression of CTGF and Invasiveness of Human Lung Adenocarcinoma CancerCell Lines

To explore the role of CTGF in invasiveness of lung adenocarcinomacells, expression of CTGF and the invasion ability in a panel of celllines, CL1-0, CL1-3, CL1-5, A549, H928, and NICH520 are examined.

Cell Culture: Lung adenocarcinoma cells are grown in RPMI 1640 medium(Life Technologies, Inc. [GIBCO BRL], Rockville, Md.) with 10% fetalbovine serum (Life Technologies, Inc.) and 2 mM L-glutamine (LifeTechnologies, Inc.) at 37° C. in a humidified atmosphere of 5% CO₂ and95% air. Lung adenocarcinoma cell lines CL1-3 and CL1-5 are sublinesthat are selected from parental CL1-0 cultures with a Matrigel-coatedpolycarbonate membrane (Collaborative Biomedical, Becton DickinsonLabware, Bedford, Mass.) in a Transwell invasion chamber as describedpreviously (Chu, Yi-Wen et al., Am. J. Respir: Cell Mol. Biol. 1997;17:353-60). Other lung adenocarcinoma cell lines used, A549, H928, andNICH520, are obtained from the American Type Culture Collection(Manassas, Va.). Adherent cells are detached from the culture disheswith trypsin/EDTA (Sigma, Deisenhofen, Germany).

Western Blotting: Cells are washed with phosphate-buffered saline (PBS)containing 5 mM EDTA and 1 mM sodium orthovanadate, and then scrapedinto lysis buffer (20 mM Tris-HCl [pH 8.0], 137 mM NaCl, 10% glycerol, 2mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 μM leupeptin, and 0.15U/mL aprotinin) and stored for 30 minutes on ice. The lysed cells arecentrifuged at 14,500 g for 30 minutes at 4° C., and the supernatant iscollected. Proteins in the supernatant are quantified byspectrophotometry. Proteins in the total cell lysate (40 μg of protein)are separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis in 10-12% gels and electrotransferred to apolyvinylidene difluoride membrane (Immobilon-P membrane; MilliporeCorp., Bedford, Mass.). After the blot is blocked in a solution of 5%skim milk, 0.1% Tween 20, and PBS, membrane-bound proteins are probedwith primary antibodies against CTGF, CYR61, β-actin. The membrane iswashed and then incubated with horseradish peroxidase-conjugatedsecondary antibodies for 30 minutes. Antibody-bound protein bands aredetected with enhanced chemiluminescence reagents (Amersham PharmaciaBiotech) and photographed with Kodak X-Omat Blue autoradiography film.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR): Reversetranscription (RT) of RNA isolated from cells is performed in a finalreaction volume of 20 μL containing 5 μg of total RNA in Moloney murineleukemia virus reverse transcriptase (MMLV) buffer (Promega, Madison,Wis., USA), which consists of 10 mM dithiothreitol, all fourdeoxynucleoside 5′-triphosphates (dNTPs; each at 2.5 mM), 1 μg of(dT)₁₂₋₁₈ primer, and 200 units of MMLV reverse transcriptase (Promega,Madison, Wis., USA). The reaction mixture is incubated at 37° C. for 2hours, and the reaction is terminated by heating at 70° C. for 10minutes. One microliter of the reaction mixture is then amplified bypolymerase chain reaction (PCR) with the following primers: CTGF primer(SEQ ID NO: 3), to produce a 500-base-pair (bp) fragment of the CTGFgene; Cyr61 primer (SEQ ID NO: 4), to produce a 450-bp fragment of theCyr61 gene; and β-actin primer (SEQ ID NO: 5), to produce a 320-bpfragment product of the β-actin gene, use as the internal control. ThePCR amplification is carried out in a reaction buffer containing 20 mMTris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, all four dNTPs (each at 167μM), 2.5 units of Taq DNA polymerase, and 0.1 μM primers. The reactionsare performed in a Biometra Thermaoblock (Biometra Inc. Florida, USA)with the following program: denaturing for 1 minute at 95° C., annealingfor 1 minute at 58° C., and elongating for 1 minute at 72° C. for atotal of 23 cycles; the final extension takes place at 72° C. for 10minute. Equal volumes of each PCR sample are subjected toelectrophoresis in a 1% agarose gel, which is then stained with ethidiumbromide and photographed under UV illumination.

Boyden Chamber Assay: For invasion assays, modified Boyden chambers areused with filter inserts (pore size, 8 μm) coated with Matrigel (40 μg;Collaborative Biomedical, Becton Dickinson Labware) in 24-well dishes(Nucleopore Corp., Pleasanton, Calif.). Approximately 2.5×10⁴ to 1×10⁶cells in 100 μL of complete RPMI 1640 medium are placed in the upperchamber, and 1 mL of the same medium is placed in the lower chamber.After 48 hours in culture, cells are fixed in methanol for 15 minutesand then stained with 0.05% crystal violet in PBS for 15 minutes. Cellson the upper side of the filters are removed with cotton-tipped swabs,and the filters are washed with PBS. Cells on the underside of thefilters are viewed and counted under a Leica Microsystems (Type:090-135.001). Each clone is plated in triplicate in each experiment, andeach experiment is repeated for at least three times.

Statistical Analysis: Statistical evaluation of the data is performedwith a two-tailed Student's t test for simple comparison between twovalues when appropriate. All statistical analyses are performed with theSPSS program package, version 10.0 (SPSS Inc., Chicago, Ill). Pearson χ²tests and Student's t tests are used to compare the clinicopathologiccharacteristics of tumors (and patients) with high and low expression ofCTGF. Survival is analyzed by the Kaplan-Meier method, and the log-ranktest is used to test the difference in relapse time and survival betweenpatients with tumors that have high and low expression of CTGF. Themedian survival times with 95% confidence intervals are calculated asdescribed. Multivariable analyses with the Cox proportional hazardsmodel are used to estimate the simultaneous effects of prognosticfactors on survival. After confirmation that the data meet theassumptions for a proportional hazards analysis, the stepwise selectionis used. Variables are retained in the model if the associated two-sidedP values are 0.10 or less. All statistical tests are two-sided. P valuesof less than 0.05 are considered statistically significant.

As a result, RT-PCR shows that CTGF mRNA is highly expressed in CL1-0cells that have low invasive and low metastatic ability, but is almostnot detectable in highly invasive CL1-3 and CL1-5 cells (FIG. 1A, upperright panel). Boyden chamber asay shows that the invasive ability ofCL1-3 and CL1-5 is fourfold to sixfold higher than that of CL1-0 cells(FIG. 1A, lower panel). The level of Cyr61mRNA, another member of theCCN family, is the same in cell lines with low or high invasive ability(FIG. 1A, upper right). Western blotting reveals that CL1-0 cellsexpress a high level of CTGF protein, and CL1-3 and CL1-5 cells expressextremely low levels of CTGF protein (FIG. 1A, upper left panel); thelevel of Cyr61 protein appears constant in CL1-0, CL1-3, and CL1-5 cells(FIG. 1A, upper left panel). Other lung adenocarcinoma cell lines (A549,NICH520, and H928) are tested to determine whether this relationshipbetween the level of CTGF and invasive ability is also present. A549cells are more invasive than NICH520 and H928 cells (FIG. 1B, lower).The highly invasive A549 cells express a very low or non-detectablelevel of CTGF protein, whereas the less invasive NICH520 and H928 cellsexpress high levels of CTGF protein (FIG. 1B, upper panel). Thus,expression of CTGF is inversely associated with an invasive and/ormetastatic phenotype of lung adenocarcinoma cell lines.

EXAMPLE 2

Overexpression of CTGF Suppresses Invasion and Metastasis in vitro

To clarify the direct role of CTGF expression in the invasiveness oflung adenocarcinoma cells, human CTGF cDNA expression vectors or controlvectors are transfected into the highly invasive CL1-5 cells. After G418selection, two single clones (CL1-5/CTGF-3 and CL1-5/CTGF-10) areisolated, a clonal mixture (CL1-5/CTGF-M), and vector control clone(CL1-5/neo), and then the levels of CTGF expression are assessed ineach.

Construction of CTGF/wt Expression Plasmids: Total RNA is extracted fromCL1-0 cells, and CTGF cDNA is cloned and amplified by RT-PCR with theprimers of SEQ ID NO: 6 and SEQ ID NO: 7 (PubMed serial numberXM-037056), and subcloned into a pcDNA3/V5-His TOPO TA vector(Invitrogen, San Diego, Calif.). The CTGF-expressing vector CTGF/wt isused in transient and stable transfections of human lung adenocarcinomacells in vitro.

Plasmid and Transient Transfection: The CTGF expression vectors aretransiently transfected into CL1-5 and A549 cells with the TransFast™transfection reagents (Promega, Madison, Wis., USA). Briefly, 3 μg ofplasmid DNA (CTGF/wt) and 8 μg of transfection reagents are mixed, andthe transfection protocol is carried out according to the manufacturer'sinstructions (Promega, Madison, Wis., USA). One hour after transfection,the cells are cultured in normal complete medium for another 8 hours.The transfected cells are harvested and subjected to an invasion assayand Western blot analysis.

Stable-Transfected Clone Selection: Purified plasmid DNA (3 μg) istransfected into CL1-5 and A549 cells with the TransFast™ transfectionreagent (Promega, Madison, Wis., USA). Twenty-four hours aftertransfection, stable transfectants are selected in Gentamicin (G418;Life Technologies, Inc.) at a concentration of 600 μg/mL. Thereafter,the selection medium is replaced every 3 days. After 2 weeks ofselection in G418, clones of resistant cells are isolated and allowed toproliferate in medium containing G418 at 100 μg/mL. Integration oftransfected plasmid DNA is confirmed by RT-PCR and Western blotanalysis.

In Vitro Cell Growth Assay: To rule out the possibility that the effectof CTGF on in vitro cell invasiveness caused by different proliferationrates among the cell lines, growth rates of CTGF-overexpressing cellsand that of the corresponding vector control cells are compared. Controlvector (pcDNA3), or the respective CTGF/wt expressing vector is added to6-cm dishes initially containing 10⁵ cells per well. At regularintervals, cells are trypsinized and resuspended, and the cell numbersare counted by hemocytometer.

CTGF-overexpressing cell lines express 3.6-fold to 5.5-fold more CTGFprotein than vector control cells (FIG. 2A). Invasive capacity is muchlower in CTGF-transfected CL1-5 cells than in CL1-5/neo control cells(FIG. 2B) (percentage of invasive CL1-5/neo control cells: CL1-5/CTGF-Mcells is 52.7% [95% CI is 23.9% to 70.8%], CL1-5/CTGF-3 cells is 44.0%[95% CI is 47.0% to 65.0%], and CL1-5/CTGF-10 cells is 54.0% [95% CI is36.9% to 55.2%]; all P<0.001). Highly invasive A549 cells are alsotransfected with the CTGF expression vector or the control vector, andthe invasive ability of the transfected cells is also examined. Aftertransfection and selection in G418, a single clone (A549/CTGF) and avector control clone (A549/neo) are isolated, and their expression ofCTGF protein is assessed by Western blotting. CTGF-transfected A549cells (A549/CTGF) clearly express CTGF protein (FIG. 3, upper), and theinvasive ability of CTGF-overexpressing A549 cells is only about 40% ofthe control A549/neo cells (percentage of invasive A549/neo cells:A549/CTGF cells is 37.7% [95% CI is 37.0% to 87.7%]; P<0.001) (FIG. 3,lower).

The growth curves for the vector control cells CL1-5/neo and theCTGF-overexpressing cells CL1-5/CTGF-M, CL1-5/CTGF-3, and CL1-5/CTGF-10are similar (FIG. 4), as are those for the vector control cells A549/neoand CTGF-overexpressing cells A549/CTGF-M (data not shown). Thus, thedecreased invasiveness of these CTGF-transfected cells is apparently notcaused by decreased proliferation rates.

EXAMPLE 3

Truncated CTGF Expression in Transfected CL1-5 Cells and Invasiveness ofthe Cells

CTGF and other CCN proteins have a structure with several domains ormodules. To determine which module participates in the inhibition ofinvasion, a series of CTGF deletion constructs are produced andcharacterized. Because CTGF is a secretory protein, the signal sequenceat the amino-terminal end is retained and sequences at thecarboxyl-terminal end are sequentially deleted.

Construction of CTGF/wt Expression Plasmids and CTGF Deletion Mutants:The CTGF-expressing vector CTGF/wt is prepared according to theprocedure in Example 2, and other three serial deletion mutants of CTGFare generated by deleting the CT domain (SEQ ID NO: 2); the CT and TSP-1domains; or the CT, TSP-1, and VWC domains. These constructs aredesignated CTGF/d3, CTGF/d2, or CTGF/d1, respectively (FIG. 5A).Deletion constructs are generated with the reverse primer SEQ ID NO: 8in combination with the forward primers SEQ ID NO: 9, SEQ ID NO: 10, andSEQ ID NO: 11, respectively. The products are subcloned into a pcDNA3expression vector, and isolated clones are designated wt, d3, d2, andd1. The invasive capacities of transiently transfected CL1-5 cellsexpressing the full-length CTGF (CTGF/wt) or its deletion mutantsCTGF/d3, CTGF/d2, or CTGF/d1 are determined according to described inExample 1.

Invasion assay shows that CL1-5 cells express CTGF/d3, which lacks theCT module, has a high invasive capacity that is equivalent to CL1-5cells expressing the control vector (FIG. 5B). The mean number ofinvasive control CL1-5/neo cells and CL1-5 cells transfected withexpressing plasmid CTGF/d3, a deletion of the CT module are 148 cellsand 385 cells (difference is 237 cells, 95% CI is 208 to 266 cells;P<0.001), respectively. CL1-5 cells transiently transfected with CTGF/d2or CTGF/d1 also have a relatively high invasive capacity compared withvector control cells.

EXAMPLE 4

Overexpression of CTGF Suppresses Invasion and Metastasis in vivo

Experimental Metastasis: Control vector- or CTGF-transfected tumor cells(CL1-5/CTGF-M or A549/CTGF-M) are prepared according to the proceduresin Example 2, and cells are washed and resuspended in PBS. Subsequently,a single-cell suspension containing 10⁶ cells in 0.1 mL of PBS isinjected into the lateral tail vein of 6-week-old male SCID mice(supplied by the animal center in the College of Medicine, NationalTaiwan University). Each clonal cell line is injected into 10 mice. Themice are killed after 8 weeks. (A preliminary study in this animal modelindicates that CL1-5 cells develope numerous lung metastasis nodules by8 weeks.) All organs are examined for metastasis formation. The lungsare removed, weighed, and fixed in 10% formalin. The number of lungtumor colonies is counted under a dissecting microscope. Representativelung tumors are removed, fixed, and embedded in paraffin. Embeddedtissue is sectioned into 4-μm sections, and the sections are stainedwith hematoxylin and eosin for histologic analysis. All animal work isperformed under protocols approved by the Institutional Animal Care andUse Committee (IACUC) of the College of Medicine, National TaiwanUniversity.

Results from FIG. 6A reveals that mice injected with CL1-5/neo orA549/neo control clones have numerous large lung metastases, whereasmice injected with CL1-5/CTGF-M or A549/CTGF-M cells have fewer andsmaller metastatic nodules in the lung. Metastatic tumors formed in thelungs by CL1-5/neo cells have the morphology of a typical adenocarcinoma(FIG. 6B). Mice injected with CL1-5/neo cells have 77.5 metastatic lungnodules, and mice injected with CL1-5/CTGF-M have 12.3 metastatic lungnodules (difference is 65.2 nodules, 95% CI is 48.9 to 81.6 nodules;P<0.001). Mice injected with A549/neo have 61.3 metastatic lung nodules,and mice injected with A549/CTGF-M have 21.7 metastatic lung nodules(difference is 39.6 nodules, 95% CI is 23.4 to 56.0 nodules; P is0.003). Thus, overexpression of CTGF in CL1-5 and A549 cells suppressesthe ability of these cells to form metastatic nodules in the lungs.

The weight of lungs from mice injected with CL1-5/CTGF cells is 390 mgand that from mice injected with CL1-5/neo vector control cells is 448mg (difference is 32 mg, 95% CI of the difference is 24 to 91 mg;P=0.005). The weight of lungs from mice injected with A549/CTGF cells is473 mg and that from mice injected with A549/neo vector control cells is617 mg (difference is 81 mg, 95% CI of the difference is 39 to 247;P=0.008). Thus, lung weight is decreased 13% in mice injected withCL1-5/CTGF cells and decreased 23.3% in mice given A549/CTGF cells,compared with lung weight in mice given vector control cells. Data fromthese two experiments are summarized in Table 1.

EXAMPLE 5

Association of CTGF Protein Expression with Lung Adenocarcinoma TissueStage, Tumor Status, Lymph Node Status, and Survival of Patients withLung Cancer

To investigate the involvement of CTGF in the progression of human lungadenocarcinoma, normal and tumor specimens from patients with lungadenocarcinoma are analyzed immunohistochemically for the expression ofCTGF.

Patients and Specimens: Lung adenocarcinoma specimens are obtained froma total of 78 consecutive patients who undergo surgical resection at theNational Taiwan University Hospital from Sep. 1, 1993, to Aug. 31, 1997.Patients who have previous history of cancers or have been treated withneoadjuvant chemotherapy and/or radiation therapy are not included fromthis study. Only lung adenocarcinoma specimens are included in thisstudy Paraffin-embedded, formalin-fixed surgical specimens are collectedfor immunohistochemical staining for CTGE The group consists of 39 menand 39 women with an age of 62±11 years (mean±standard deviation).Written informed consent is obtained from all patients. The histologicidentification of lung cancer is determined as recommended by the WorldHealth Organization. Tumor size, local invasion, and lymph nodemetastasis are determined at pathologic examination. The final diseasestage is determined by a combination of surgical and pathologicfindings, according to the current tumor-node-metastasis system for lungcancer staging. Follow-up data are obtained from the patients' medicalcharts and from our tumor registry service. The survival time ofpatients is calculated from the date of operation to the date of death.The relapse time is calculated from the date of operation to the date oflocal recurrence or distant metastasis. Median follow-up is 37.4 months(ranging from 1 to 117 months).

Immunohistochemistry: After rehydration, sections (4 μm) of aparaffin-embedded tissue block that has been cut on glass slides areincubated in 3% hydrogen peroxide to block endogenous peroxidaseactivity. After trypsinization, the sections are blocked by incubationin 3% bovine serum albumin (BSA) in PBS. The primary antibody, apolyclonal goat anti-human CTGF antibody (R&D Systems, Minn.), areapplied to the slides at a dilution of 1:50 and incubated at 4° C.overnight. After washes in PBS, the samples are treated withbiotin-labeled secondary antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.), at a dilution of 1:500 to 1:250 for 1 hour at room temperature.Detection is performed with an ABC-kit (DakoCytomation, Denmark). Theslides are stained with diaminobenzidine (DAB), washed, counterstainedwith Delafield's hematoxylin, dehydrated, treated with xylene andmounted. A scoring system is devised to assign a staining intensityscore for CTGF expression from 0 (no expression) to 3 (highest intensitystaining). Immunostaining is classified into one of two groups accordingto both intensity and extent: low expression is defined as no stainingpresent (staining intensity score=0) or positive staining detected inless than 10% of the cells (staining intensity score=1) and highexpression is defined as positive immunostaining is present in 10%(staining intensity score=2) or more of the cells (staining intensityscore=3).

Expression of CTGF protein is high (intensity level 3) in the normallung epithelium (FIG. 7A) and moderate to high (intensity level 2 to 3)in stage 1 lung adenocarcinoma cells (FIG. 7B). In these tumors, theprotein is predominately localized to the cytoplasm. Expression of CTGFis reduced (intensity level 1) in low-grade metastatic epithelial tumorcells (FIG. 7C). CTGF is not detected in adenocarcinoma cells but isdetected in the normal fibroblast and epithelial components of the samefield, which are used as the corresponding positive staining control(FIG. 7D).

Table 2 also illustrates the level of CTGF protein associated with someprognostic factors for lung adenocarcinoma. The patients with lungadenocarcinoma are divided into two groups, those with tumors expressinglow levels of CTGF (level 0 and 1) and those with tumors expressing highlevels of CTGF (level 2 and 3). The low expression of CTGF is found tobe statistically significantly associated with higher grade lymph nodemetastasis (N1-3 vs. N0, P=0.014), larger tumor size (T2-4 vs. T1,P=0.031), and advanced-stage disease (stage III-IV vs. stage I-II,P=0.001). The median time to postoperative disease recurrence is longerfor patients whose tumors express a high level of CTGF (28.8 months; 95%CI is 8.0 to 49.6 months) than for patients whose tumors express a lowlevel of CTGF (10.0 months; 95% CI is 3.3 to 16.7 months) (two sidedlog-rank test, P=0.07; FIG. 8A). The 5-year disease-free survival rateis 35% (95% CI is 15% to 54%) in the high expression group and 22% (95%CI is 10% to 33%) in the low expression group. In addition, the mediansurvival time is longer for patients whose tumors express a high levelof CTGF (66.7 months; 95% CI is 47.9 to 85.6 months) than for patientswhose tumors express a lower level of CTGF (18.2 months; 95% CI is 5.2to 31.1 months) (difference is 48.5 months, 95% CI is 33.5 to 63.5months; two sided log-rank test, P=0.02, FIG. 8B). The 5-year overallsurvival rate is 54% (95% CI is 34% to 74%) in the high expression groupand 26% (95% CI is 14% to 38%) in the low expression group. Thus, theexpression of CTGF is statistically significantly associated with lymphnode metastasis and overall survival.

EXAMPLE 6

Invasion Capacity of CRC Cell Lines is Inversely Related to CTGFExpression in vitro

To explore whether CTGF affects the invasiveness of human CRC(colorectal cancer) cells, the expression level of CTGF in four humancancer cell lines, HCT116, COLO205, HT-29, and Caco-2, and a mouse cellline CT26 are examined by RT-PCR and Western blotting assay. Also,invasive ability of the cells is assayed by Boyden chamber assay.

Cell culture: HCT116 and Caco-2 cells are maintained in DMEM (LifeTechnologies, Inc.), with the addition of 4 mM L-glutamine and 10 mMsodium pyruvate (Sigma Chemical Co., St. Louis, Mo.). In addition, themedium used for Caco-2 cells is supplemented with 10 μg/ml transferrin.COLO205 and HT-29 cells are cultured in RPMI 1640 medium (LifeTechnologies), and the medium used for CT26 cells contains an additional10 mM HEPES, 4.5 g/L glucose and 10 mM sodium pyruvate. All media usedfor cell culture are supplemented with 10% fetal bovine serum and a 1%penicillin (10,000 units/ml)-streptomycin (10,000 mg/ml) solution (LifeTechnologies, Inc.). Cells are maintained at 37° C. in the presence of5% CO₂ in air. All cells are passaged into new medium every 2-3 days andbefore confluence.

Western blotting, RT-PCR, and Boyden chamber assay are performedaccording to what is described in Example 1.

Results from FIG. 9A show that HCT116 cells have the lowest mRNA (FIG.9A, lower) and protein levels (FIG. 9A, upper) of CTGF. HT-29 andCOLO205 cells exhibit moderate level of CTGF mRNA and protein. CT26 andCaco-2 cells have the strongest expression level of CTGF. Invasion assayof the cells reveals that HCT116 cells display the highest invasivenessamong these cells; in contrast, Caco-2 and CT26 cells show only weakinvasive ability (FIG. 9B). The above results demonstrate that invasiveability is inversely correlated with the level of CTGF expression inhuman CRC cell lines.

EXAMPLE 7

Altered Level of CTGF Affects the Invasiveness of Human CRC Cell Lines

To investigate the direct role of CTGF in the invasion and metastasis ofhuman colorectal cancer, sense and antisense CTGF-expressing vectors aretransfected into HCT116 and CT26 cells, respectively. After G418selection, CTGF-overexpressing HCT116 (HCT116/CTGF), anti-senseCTGF-overexpressing CT26 (CT26/AS-CTGF) transfectants and Neo controlcells are analyzed for their expression level of CTGF mRNA and protein.

Plasmid, Transient Transfection, and Reporter Gene Assay: The CTGF(sense) expression vectors are transiently transfected into HCT116 cellswith TransFast™ transfection reagents (Promega, Madison, Wis., USA).Other procedures are as described in Example 2.

Generation of HCT116 and CT26 cell lines that constitutively expresssense and antisense CTGF: HCT116 cells expressing CTGF (sense) or CT26cells expressing CTGF (antisense) are established by transfection withthe expression vectors, CTGF (sense) or CTGF (antisense). After 48 h oftransfection, cells are trypsinized and replated in RPMI 1640 with 10%fetalcalf serum and 1000 μg/ml G418. G418-resistant clones are selectedand expanded.

FIG. 10A shows that HCT116/CTGF cells exhibit a 4-5 fold increase ofCTGF mRNA and protein as compared to HCT116/Neo cells. Interestingly,the invasive capacity of HCT116/CTGF cells is dramatically reduced to20% of that of HCT116/Neo cells (FIG. 10B). Furthermore, the anti-senseCTGF transfection experiment shows that the endogenous CTGF mRNA andprotein levels are effectively diminished in CT26/AS-CTGF cells (FIG.10A). In a Boyden chamber assay, the invasive ability of CT26/AS-CTGFcells is increased about 3-4 fold over than that of CT261Neo cells (FIG.10B). This clearly shows that increased CTGF expression level in CRCcells result in inhibition of their invasive ability. To rule out thepossibility that the effect of CTGF on in vitro cell invasiveness causedby different proliferation rates among these cell lines, growth rates ofHCT116/CTGF, CT26/AS-CTGF, and their Neo control cells are compared. Thegrowth rate of these cells is the same, suggesting that the alteredinvasive ability of those transfectants can not be attributed to theirdifferent growth rate (data not shown).

EXAMPLE 8

Reduced CTGF Expression in CT26 Cell Line Increases Liver Metastasis invivo

To further clarify whether modulation of CTGF expression affects themetastasis activity of colorectal cancer cells in vivo, BALB/c mice aregiven an intrasplenic/portal injection of CT26/AS-CTGF and CT26/Neocells.

Experimental metastasis: CT26 cells or transfectants are washed, and5×10⁵ cells are suspended in 1 ml of PBS. Syngeneic 6-8 week-old BALB/cfemale mice are injected with 0.1 ml of these cells into the portal veinvia intrasplenic injection, which is performed by a minilaparotomy (0.5cm in length) over the left flank. Autopsies are performed when animalsare moribund or after 6 weeks with macroscopic and microscopicexamination for the presence of metastasis. These experiments areapproved by the institute's Animal Welfare Committee.

Most mice injected with CT26/AS-CTGF cells become moribund within 4weeks. CT26/AS-CTGF cells form intrasplenic tumors to the same extentand frequency as compared with CT26/Neo cells (FIG. 11). However, thenumber of hepatic metastatic nodules is significantly increased in miceinjected with CT26/AS-CTGF as compared with CT26/Neo cells (FIG. 11, andTable 3; P=0.0039). The liver weight is also remarkably increased forthose injected with CT26/AS-CTGF cells but not for those injected withthe neo control cells (Table 3, P=0.0008). Histopathological examinationof the metastatic tumors formed in the livers shows that CT26/AS-CTGFcells have an uneven invasive front, with vascular emboli (arrow) in theadjacent liver parachyma (FIG. 12, III and IV). In contrast, CT26/Neocells have a smooth, pushing border (FIG. 12, I and II). The aboveresults demonstrate that CTGF acts as a crucial negative regulator ofhepatic metastasis by CRC in mice.

EXAMPLE 9

CTGF is an Independent Prognostic Factor in Patients with CRC

To investigate the involvement of CTGF in the progression, normal andtumor specimens are analyzed immunohistochemically for the expression ofCTGF, and a CTGF-specific antibody is used to determine the expressionlevel of CTGF in CRC. This antibody does not cross-react with other CCNmembers.

Patients: Our study includes 119 consecutive patients with CRC treatedat National Taiwan University Hospital between December 1996 and July1999. They are made up of 61 male and 58 female CRC patients. Theiraverage age is 62.7±13.4 years (median 63, ranging 37-89 years). Allpatients undergo complete surgical resection, and their clinical andpathological data are available. Patients with familial adenomatouspolyposis, hereditary non-polyposis CRC syndrome (according to Amsterdamcriteria), inflammatory bowel diseases or who have had a malignant tumorwithin 5 years are excluded from this study. Tumor stage is based on thepost-operative pathological report and a pre-operative clinicalevaluation including chest radiogram, carcinoembryonic antigen (CEA)level and abdominal ultrasonography or computed tomography. Informationabout clinical outcome is obtained from a hospital chart review or adirect telephone interview with the patient's personal physician. Allpatients are followed up and this involved periodic examinationscomprising serum blood-chemistry panels, CEA level, endoscopy andabdominal ultrasonography and radiograms of the thorax. Computedtomography or magnetic resonance imaging is also performed in caseswhere there is a suspected tumor recurrence. The overall survival timeis calculated from the date of surgery to the time of the last visit ordeath and the disease-free survival time from the date of resection torelapse. The median follow-up time is 58.9 months. Tumor distributionaccording to primary site is 29 in right colon, 53 in the left colon(from splenic flexture to end of sigmoid colon) and 37 in the rectum.Fourteen patients have stage I, 37 have stage II, 51 have stage III and17 have stage IV disease. The 5-year survival rates are 90%, 74.5%,55.0% and 5.9% for stage I to IV, respectively.

Immunohistochemistry: All procedures are the same as described inExample 5 except samples are from normal colon epitheliums, colon polypspecimens, non-invasive colorectal tumors, and metastatic colorectaltumors. The results of immunohistological staining are classified usingintensity; these are level 0 (negative staining), intensity level 1 (<5%of tumor cell stained), intensity level 2 (<50% of tumor cells stained),and intensity level 3 (>50% of tumor cells stained).

The results of the immunohistological staining are classified asintensity level 0 (negative staining), intensity level 1 (<5% of tumorcell stained), intensity level 2 (<50% of tumor cells stained), andintensity level 3 (>50% of tumor cells stained). As shown in FIG. 13,high level of immunoreactivity for CTGF (intensity level 3) is detectedin normal colon epithelium (FIG. 13A) and a colon polyp specimen (FIG.13B). The CTGF protein is predominantly localized in the cytoplasm orthe membrane of normal or tumor epithelial cells. Interestingly, highlydifferentiated and non-invasive colorectal tumors also show a highexpression level for CTGF (FIG. 13C). In contrast, very weakimmunoreactivity (intensity levels 0 and 1) for CTGF is observed in poordifferentiated and metastatic colorectal tumors (FIG. 13D). Negativestaining for CRC is showed when immunostaining using an IgG control(FIG. 13A, inset). Of the 119 CRC analyzed, low expression level(intensity levels 0 and 1) and high expression level (intensity levels 2and 3) of CTGF in CRC specimens is 44% (53 of 119) and 56% (66 of 119),respectively. The incidence of low CTGF expression among the four TNMstages is 29% (4 of 14) in stage I, 35% (13 of 37) in stage 2, 47% (24of 51) in stage III and 71% (12 of 17) in stage IV disease (P=0.059).The relationship between the level of CTGF expression and theclinicopathological characteristics are summarized in Table 3. Nosignificant relationship is found between the level of CTGF expressionand age of patients, their sex, tumor stage, tumor site, grade ofdifferentiation, preoperative CEA level, and invasion depth. Colorectaltumors with low CTGF expression are more often detected as lymph nodemetastasis (P=0.044). Furthermore, CTGF expression is associated withdisease outcome, that is, patients with low CTGF expression have theshorter survival (P<0.001) and more frequent recurrence (P<0.001)compared to CRC patients with high CTGF expression.

Patients are stratified by tumor stage, stage II and III, and patientswith high CTGF expression show a better survival rate and recurrent-freeadvantage compared to those with low CTGF expression (FIG. 14A and B).Influence of each clinicopathological characteristic and the expressionpattern of CTGF on patient survival and disease-free status of stage IIand III patients is analyzed using the extended Cox regression model andthe results are listed in Table 4. Among the stage 2 and 3 tumors,patients whose tumors have a high CTGF expression also have a betterdisease-free status (hazard ratio (HR)=0.143, P<0.001) and a betteroverall survival (HR=0.186, P<0.001) than those who have a low CTGFexpression level. The data suggests that it is possible that invasiondepth of T4 might increase the risk of recurrence (HR=3.72, P=0.04).CTGF expression is the most significantly independent predictor in thisunivariate model analysis and no other variables show a relationship tothe disease outcome in the series of patients examined (Table 4). The5-year survival rate for stage II disease patients with tumors showinghigher CTGF expression is 87.5% but those with a lower CTGF expressionis 47.5% (P=0.0052). In particular, the 5-year survival rate for stageIII disease patients with tumors showing higher CTGF expression is 76.7%but those with a lower CTGF expression is only 28.1% (P=0.005). Similarresults are obtained when the disease-free time is the end point, thatis, patients whose tumors show a lower expression of CTGF, recurrenceoccurs earlier. Of the 51 patients with high CTGF expression in stage IIand III CRC, only 7 (13.7%) develop tumor recurrence (6 distalmetastasis and 1 loco-regional). In contrast, 24 of the 37 (64.9%)patients whose tumors have a low CTGF expression develop tumorrecurrence (20 distal metastases and 4 loco-regional). A significantdifference in 5-year survival between the patients with (77.9%) andwithout (8.0%) recurrence is noted (P<0.001). Taken together, all datastrongly suggest that CTGF acts as an independent prognostic factor forCRC patients, especially for stages II and III tumors. Since the deathof CRC patients is normally due to early metastasis, CTGF plays a rolein modulating the invasiveness and metastasis of CRC. TABLE 1Antimetastatic effect of connective tissue growth factor (CTGF) in thelung metastasis model in SCID mice* Lung metastasis Nodule Weight MedianNo. Median, mg Cell line (range) P value† (range) P value† CL1-5/neo77.5(69-91) 448(430-480) CL1-5/CTGF 12.3(11-13) <0.001† 390(380-410)<0.005† A549/neo 61.3(54-73) 617(600-640) A549/CTGF 21.7(19-24)  0.003†473(420-500)  0.008†*Each group contains 20 moce. Statistical evaluation of the data isperformed with a Student's t test. Test for simple comparison betweentwo values is also used when appropriate. All statistical tests aretwo-sided.†P values of less than 0.05 are considered statistically significant(CL1-5/neo versus CL1-5/CTFG; A549/neo versus A549/CTGF).

TABLE 2 Clinicopathologic characteristics of tumors with high and lowexpression of connective tissue growth factor (CTGF) protein Low CTGFHigh CTGF expression expression No. P Characteristic 54 24 value Age, y(mean ± standard 611.5 ± 11.0 62.0 ± 12.0 0.47* deviation) Sex, No. ofpatients Male 31 8 0.05 Female 23 16 Stage, No. of patients I and II 1918 0.001 III and IV 35 6 Tumor status, No. of patients T1 10 10 0.031T2-4 44 14 Lymph nodal status, No. of patients† N0 22 17 0.014 N1-3 32 7*P value for age is derived with a two-sided Student's t test; other Pvalues are derived with a two-sided Pearson chi-square test.†The tumor stage, tumor status, and lymph node status are classifiedaccording to the international system for staging lung cancer.

TABLE 3 Clinical and pathological characteristics for high and low CTGFexpression in colorectal cancer (CRC) High-CTGF Low-CTGF No.Characteristics 66 53 P-value Age (years, mean ± 61.7 ± 13.7 61.8 ± 13.00.889 standard deviation) Sex Male 38 23 0.143 Female 28 30 Tumorside^(δ) Right 14 15 0.247 Left 34 19 Rectum 18 19 Tumor Poor 4 3 0.158differentiation Moderate 58 40 High 4 9 CEA^($) level (ng/ml) ≦3 33 250.854 >3 33 28 Stage I 10 4 0.059 II 24 13 III 27 24 IV 5 12 Lymph node0 40 22 0.044* ≧1 26 31 Invasion depth T1 1 3 0.278 T2 13 5 T3 50 39 T42 3 Intra-tumor Present 21 18 0.846 invasion^(Φ) Absent 45 35 RecurrenceNo 59 28 <0.001* Yes 7 25 Vital status Alive 53 17 <0.001* Dead 13 36*Statistical significance (P < 0.05).^(δ)Tumor side. Right: cecum to splenic flexture; Left: splenic flextureto sigmoid colon.^($)CEA: carcinoembryonic antigen.ΦIntra-tumor invasion (present): if pathologic report reveals one ofvenous, lymphovessel or perinurial invasions.

TABLE 4 Predictors^(#) for mortality or recurrence in colorectal cancerof stage II or III Overall survival Disease-free Hazard HazardCharacteristic Category ratio 95% CI P value ratio 95% CI P value SexMale vs. Female (ref) 1.39 0.7-2.8 0.36 1.34 0.6-2.7 0.42 Age(years) >65 vs. ≦65 (ref) 1.66 0.5-5.1 0.37 1.41 0.5-4.3 0.54 Tumor sideRight (ref) vs. left 0.53 0.2-1.3 0.171 0.58 0.2-1.4 0.21 vs. rectum1.12 0.5-2.6 0.794 0.89 0.4-2.1 0.79 CEA (ng/ml) ≦3 (ref) vs. >3 1.020.5-2.1 0.96 1.10 0.5-2.2 0.80 Tumor differentiation Well (ref) vs.Moderate 0.94 0.2-4.1 0.93 0.89 0.2-3.8 0.87 vs. Poor 1.88 0.4-9.7 0.452.21  0.4-11.0 0.33 Stage II (ref) vs. III 1.68 0.8-3.6 0.17 1.960.9-4.2 0.08 Invasion depth T2 (ref) vs. T3 vs. 2.53  0.3-18.7 0.36 2.17 0.3-16.1 0.45 T4 (no T1) 3.72  0.2-59.6 0.35 10.97  1.1-105.9 0.04*Intratumor invasion Present vs. Absent (ref) 0.43 0.7-2.8 0.115 1.670.8-3.5 0.16 CTGF expression High vs. Low (ref) 0.143 0.06-0.34 <0.001*0.186 0.09-0.41 <0.001**Statistical significance (P < 0.05).^(#)Extended Cox regression model, univariate, with age as thetime-varying covariate.

1. A method for determining metastatic potential of tumor cells,comprising the steps of evaluating expression levels of CTGF in thecells, wherein the expression levels of CTGF is inversely associatedwith the metastatic potential of tumor cells, and the CTGF is encoded bya nucleotide of SEQ ID NO: 1, and wherein the tumor cells are colorectalcancer cells.
 2. The method according to claim 1, wherein the tumorcells express an expression levels of CTGF which is inversely associatedwith the metastatic potentials.
 3. The method according to claim 1,wherein the steps comprise evaluating expressed protein levels of CTGF.4. The method according to claim 1, wherein the steps comprisesevaluating levels of mRNA encoding CTGF.
 5. A method for determiningmetastatic potential of tumor cells, comprising the steps of evaluatingexpressed levels of CT module in a nucleotide sequence encoding CTGF inthe cells, wherein the expression levels of CTGF is inversely associatedwith the metastatic potential of tumor cells, and the CT module is SEQID NO: 2, and wherein the tumor cells are at least one selected from thegroup consisting of lung cancer cells and colorectal cancer cells. 6.The method according to claim 5, wherein the tumor cells express anexpression levels of CTGF which is inversely associated with themetastatic potentials.
 7. The method according to claim 5, wherein thesteps comprise evaluating expressed protein or polypeptide levels of theCT module.
 8. The method according to claim 5, wherein the stepscomprise evaluating levels of mRNA encoding the CT module.